Differentiation of Human Mesenchymal Stem Cells into Insulin Producing Cells by Using A Lentiviral Vector Carrying PDX1.

OBJECTIVE
Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults.


MATERIALS AND METHODS
We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions.


RESULTS
After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations.


CONCLUSION
MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation.


Introduction
Diabetes afflicts more than 300 million people in the worldwide, accounting for approximately 12% of the world's total health cost. Diabetes is classified two broad categories, type 1 (5-10%) and type 2 (90-95%) (1,2 ). Type I diabetes is an immunologically-mediated devastation of the insulin producing cells (IPCs) in the pancreatic islet (3). Although insulin therapy is an ordinary treatment for type 1 diabetes, a regimen of several daily injections and need to inject for each meal may be difficult to achieve optimal glycemic control (4). Other treatments such as transplantation of the pancreas have disadvantages; such as the lack of donors for islet transplantation and immune rejection (5). Stem cells that produce β-cells are a new promising tool.
Embryonic stem cells (ESCs) are pluripotent cells that can differentiate into three germ layers. These cells have the capability to differentiate into insulin producing cells, however they have increased the raise risk for tumor forma-tion in the recipient (6). Adult stem cells such as mesenchymal stem cells (MSCs) are multipotent cells with self-renewal capability to differentiate into ectodermal, mesodermal, and ectodermal tissues (7). Human MSCs are isolated from somatic tissues including bone marrow (BM), adipose tissue or fetal tissue, such as amniotic fluid (AF), Wharton's jelly and umbilical cord blood (8).
Pancreatic and duodenal homeobox factor 1 (PDX1) is a transcriptional factor necessary for embryonic development of the pancreas. PDX1 plays a distinct role in proliferation and differention. Heterozygote missense and frame shift mutations result in decreased secretion of insulin from β-cells which creates maturity onset diabetes of the young (MODY4) (9). Lentiviral vectors result in high transmission efficiency, stable gene expression in target cells and are useful tool for genetic manipulation (10). In this study we have used a lentiviral vector to deliver the PDX1 gene into MSCs to generate IPCs. After transduction we measured insulin and C-peptide production in these cells. INSULIN and PDX1 gene expressions were evaluated by real-time polymerase chain reaction (RT-PCR) and immune cytochemical analyses.

Cell isolation and culture analysis of human mesenchymal stem cell
We obtained three healthy samples for BM transplantation from healthy donors (age 20-40 years). These samples were obtained after patients were informed about the study and signed informed consents and permission from the local Ethics Committee at Taleghani Hospital (Tehran, Iran). BM samples (5 ml) were obtained from the posterior superior iliac crest of each patient and transferred to clean heparinized collection tubes. Samples were diluted with phosphate-buffered saline (PBS, Merck, Germany) at 1:1 ratio to which 3-4 ml Ficoll (Ficoll-paque, 1.073 g/ml, Pharmacia Uppsala, Sweden) was added. Samples were subsequently centrifuged for 20 minutes at 400 g. The mononuclear cells located at the plasma: density gradient medium interface were collected and transferred to a new tube, PBS was added to the cells tubes were centrifuged for 5 minutes at 400 g, after which the supernatant was discarded. The cells were cultured in 5 ml of Dulbecco's Modified Eagle's Medium-low glucose (DMEM-LG, Gibco, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco, USA), and 100 U/ml of penicillin/streptomycin (Gibco, USA), after which they were incubated at 37˚C in 5% humidified CO 2 . After 3-4 days, we replaced half of the medium with fresh medium to remove any non-adherent cells. When the cells reached 80-90% confluency, they were collected by treatment with 0.25% trypsin (Gibco, USA) and 1 mM ethylenediaminetetraacetic acid (EDTA) for 2-5 minutes at 37˚C. After centrifugation, MSCs were cultured in 75 cm 2 flasks until confluent.

Flow cytometry analysis of human mesenchymal stem cells
After the fourth passage, MSCs were detached by trypsinization. The cells were washed with PBS, and incubated with labeled phycoerythrin (PE)-conjugated monoclonal antibodies (Invitrogen, USA) at the dilutions recommended by the manufacturer at 4˚C for 25 minutes in the dark. The antibodies used were cluster of differentiation (CD) markers; CD90, CD34, CD105, and CD31-PE (BD Biosciences, USA). PE-labeled isotype-matched immunoglobulin was the negative control. The labeled cells were analyzed on a FACS Caliber flow cytometer (Becton-Dickinson, FACScan, San Jose, CA, USA).

Adipocyte and osteoblast differentiation of mesenchymal stem cells
MSCs at passage 4 were used for osteoblast and adipocyte differentiation. For adipocytes, cells were placed in medium containing L-DMEM (Bioidea, Iran), 10% FBS, 10 6 M dexamethasone (Sigma, USA), and 0.05 mg/dl ascorbic acid (Merck, Germany). After two weeks, differentiated adipocyte cells were stained with oil red O (Sigma, USA).

Oil-red O staining
Cells were washed with PBS and fixed with 4% para-formaldehyde (Merck, Germany) for 20 minutes, after which 60% isopropanol (Merck, Germany) was added. After 5 minutes, the isopropanol was removed. Oil red O was dissolved in 99% isopropanol and added to cells. The cells were allowed to incubate for 15 minutes at room temperature. The dye was subsequently removed and cells were washed with PBS until the water rinsed off clear.

Alizarin-red staining
The cells were washed with PBS, fixated with 4% para-formaldehyde, immersed in a 1% alizarin red solution for 10 minutes and rinsed with distilled water (DW).

Plasmids
EX-M0942-Lv105 plasmid that carried the PDX1 gene was purchased from (Genecopoeia, USA). PsPAX2 and pMD2.G vectors for viral packaging were purchased from (Invitrogen, USA). These vectors were transformed in DH5α. Plasmids were purified with a kit (real-biotech, plasmid mini kit, Taiwan). One single copy of EX-M0942-Lv105 contained 7730 bp. We used a software program available at: http://cels.uri. edu/gsc/cndna.html to calculate the copy number of the plasmid PDX1. There were 7.23×10 10 copies per µ liter. This calculation was needed for viral particle titration. Serial dilutions of the plasmid (7.23×10 7 -7.23×10 9 ) were prepared, after which we created a PDX1 plasmid standard curve for determine the copy number of integrated lentiviruses.

Production of lentiviral vector in HEK-293 cells
HEK-293T cells that had density of 4×10 6 / ml were seeded in 100 mm dishes (JET Biofil, China) that contained L-DMEM supplemented with 10% FBS. When the cells reached 80% confluency, transfection with PDX1 plasmid and the two packaging viral vectors (pMD2.G and psPAX2) was performed according to the calcium-phosphate protocol. The cells supernatants were collected every 24 hours and fresh medium that contained serum was added to the cells. After 72 hours, the total viral medium was centrifuged for 10 minutes at 2100 g and filtered through a 0.45 micron syringe filter. Virus's particles were concentrated by poly ethylene glycol (PEG, Sigma, USA) 50% and Nacl 5 M (Merck, Germany). The supernatant was incubated for 16-20 hours on a shaker at 4˚C. After which the samples were centrifuged for 10 minutes at 4100 g and a temperature of 4˚C. Sediment dissolved in centrifuged in 1 ml of DMEM-F12 medium and aliquted into sterile 1.5 µl microtubes. Cells were collected and PDX1 gene expression examined by PCR and Western blot to confirm virus production.

Titration lenti-vector by real-time polymerase chain reaction
HEK-293T cells were seeded at density of 6×10 4 /mL per well in 12-well tissue culture plates. We added 50 and 100 μl of concentrated virus to the cells. After 16 hours, the culture medium was replaced with fresh DMEM media supplemented with 10% FBS. After 96 hours, we extracted the DNA from traduced cells with a Spin Blood Mini Kit (Invisorb ® , Germany). Extracted DNA was stored at -20˚C until use. In quantitative real-time PCR (qPCR), a standard curve was generated by using 10 log that included a serial dilution (7.23×10 7 -7.23×10 9 particles/μl) of plasmid EX-M0942-Lv105 which contained the puromycin resistance gene sequence. We used the Applied Biosystems Step One software v2.2 to calculate the standard curve. Recombinant DNA (unknown sample) extracted from HEK-293 and standard samples were run on Applied Biosystems Step One (Applied Biosystems, USA) with the SYBR-Green kit (Takara, Japan). Specificities of the products were evaluated by melting curve analysis. We used primer specific for the puromycin resistance gene to quantify the copy numbers of viral particles (Table 1). qPCR values are mean ± standard error of mean (SEM).

Transduction of mesenchymal stem cells
We seeded 5×10 5 /well passage-4 human MSCs that were 70-80% confluent in a six-well plate. MSCs were transduced with medium that contained viral particles at 37˚C for 6-8 hours by the multiplicity of infection (MOI) of 50 and then replaced with fresh L-DMEM that contained 10% FBS. Medium replacement was performed two days per weeks.

Reverse transcription polymerase chain reaction and real-time polymerase chian reaction
Total RNA was extracted from transfected HEK-293 cells by Trizol (Invitrogen, USA) according to the manufacturer's instructions. Total RNA was extracted at days 7, 14 and 21 post infection from MSCs. cDNA was synthesized from the RNA template according to the instructions below. First strand cDNA was synthesized from the RNA template. A total of 50 ng RNA was reverse transcribed by random hexamer primer using high capacity cDNA Synthesis Kit (Intron, Korea) at 42˚C for 60 minutes and 70˚C for 5 minutes. PCR reaction was performed in 25 μl final volume that contained 20.75 μl PCR master mix (Vivantis, Malaysia), 0.25 μl Taq polymerase (Vivantis, Malaysia), 2 μl PDX1 forward and reverse primers (Table 1) and 2 μl cDNA. Amplification conditions were initial denaturation at 95˚C for 3 minutes and followed by 35 cycles at 60˚C and extension at 72˚C for 1 minute. Real-time PCR was performed using the Applied Biosystems Step One (Applied Biosystems, USA) with a SybrGreen Master Mix Kit (Takara, Japan) and performed in 10 μl volume that contained 1 μl cDNA and 1 μl primers (Table 1). GAPDH was used as a reference gene. The device Program was primary denaturation at 95˚C and annealing at 60˚C for 40 cycles. The qRT-PCR values are expressed as mean ± SEM of three independent real-time PCR experiments.

Western blot analysis
We used Western blot analysis to measure of PDX1 expression in transfected HEK-293. Total protein was extracted from transfected and non-transfected HEK-293 by radioimmuno precipitation assay (RIPA) buffer that contained 10 mM tris-HCl (pH=8), 1% nonyl phenoxypolyethoxylethanol (NP-40), 10% glycerol, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA and 100 mM Nacl with protease inhibitor cocktail (Roche Diagnostic, GmbH, Germany). There were 50 μg of lysates loaded into each well of a 15% SDSpolyacrylamide (SDS-PAGE, Merck, Germany) gel for electrophoresis analysis. After transfer of the separated proteins onto nitrocellulose membrane (Amersham Biosciences, USA) the membrane was blocked with 3% nonfat skim milk in a tris-buffered solution with 0.1% Tween 20 (TBST, Sigma, USA) for 2 hours, after which it was incubated overnight at 4˚C with mouse anti-human PDX1 primary antibody (#ab84987, Abcam, Cambridge, MA, USA). The membrane was washed three times with TBST, then incubated with polyclonal anti-mouse horseradish proxidase-conjugated (HRP) secondary antibody (1:500, #AP8036, Razi Biotech, Iran) for 1hour at room temperature. After washing three times with TBST, the bands were visualized using enhanced

Insulin and C-peptide secretion and content
At 14 days after induction, the cells were washed with PBS and incubated in Krebs Ringer bicarbonate (KRB) buffer without glucose for 2 hours. Then, the cells were incubated in KRB that contained 5.5, 15 and 25 mM glucose at 37˚C. After incubation, cells were centrifuged and the supernatant collected and stored at -70˚C until the assayed. In order to measure intracellular insulin and C-peptide, total protein was extracted from the cells by lysis buffer and protease inhibitor, then sonicated three times for 15 seconds each at 40 W and centrifuged at 12000 rpm for 15 minutes at 4˚C. The supernatant was collected and kept at -70˚C utile use. Insulin and C-peptide were determined by enzyme immunoassay ELISA kit (#10-1132-01, #10-1141-01, Mercodia, Uppsala, Sweden). The conversion factors for insulin were 1 μg/ml=23 mU/l, 1 mU/l= 6.0 pmol/l and for Cpeptide, 1 μg/l corresponded to 331 pmol/l.

Statistical analysis
The data were analyzed by Graph Pad Prism 5 (GraphPad, La Jolla, CA, USA). One-way ANO-VA followed by Bonferroni's post-hoc test for comparison of experimental groups with the control was used to compare results. P values <0.05 were considered statistically significant.

Cell culture and characterization of human bone marrow mesenchymal stem cells
We collected BM samples from healthy donors. Mononuclear cells were obtained through density centrifugation by using a ficoll-paque solution. Non-adherent cells were removed at 3-4 days after primary culture and adherent cells were reached at 90% confluency at day 21. Then, the cells were seeded into a plastic flask. Primary MSCs showed a characteristic spindle-shaped morphology (Fig.1A). Flow cytometry analysis showed that sub cultured MSCs expressed high levels of CD105 and CD90 and low levels of CD31 and CD34 (Fig.1B). MSCs were successfully differentiated into osteogenic and adipogenic lineages. We observed osteogenic differentiation after 21 days of culture in osteogenic media as confirmed by alizarin-red staining. Adipocytes cells were confirmed by oil-red O staining at day 14 (Fig.2). chemiluminescence (ECL) reagent (Ariyatous Biotech, Iran) according to the manufacturer's instructions.

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay
We performed the MTT assay to assess viability of MSCs during differentiation to IPCs. 10 5 transduced cells per well on days 0, 7, 14 and 21were seeded into 96-well. The cells were incubated for 24 hours at 37˚C. The supernatant was removed and freshly prepared MTT (5 mg/ml) solution was added to the cells. The cells were subsequently incubated 4 hours at 37˚C. After the incubation period, 100 μl dimethyl sulfoxide (DMSO) was added to the wells and were incubated for 20 minutes at room temperature. The optical density (OD) was read at a 570 nm wavelength. Viability compared to control cells was calculated.

Production of LV-PDX1 in HEK 293 cells
The LV-PDX1 vector was amplified and packaged in HEK-293 cells. Virus production was confirmed by detection of PDX1 in HEK-293. RNA was extracted from transfected cells and cDNA synthesized. PCR analysis was performed to examine expression PDX1 in the transfected cells (Fig.3A). We examined expression of the PDX1 protein by Western blot in transfected cells (Fig.3B).

Titration of lentivector by quantitive polymerase chain reaction (qPCR)
To perform titration by quantitative real-time PCR, plasmid EX-M0942-Lv105 that contained the puromycin resistance gene and puromycin primers was used to derive the standard curves. The number of lentiviral vector copies was measured by the standard curve was created automatically with Applied Biosystems Step One v2.0 software in each run by plotting the Ct number against the copy numbers of each standard and quantification of viral DNA for unknown samples was inferred from the regres-sion line (Fig.4A, B). Melting curve was generated to determine melting point of the products. Melting point was 93.85˚C. We observed that no contaminating products were present in this reaction (Fig.4C).

A B
A B C ited a spindle-shape morphology before differentiation (Fig.5A), initial morphological changes were observed in the transduced cells at day 5 ( Fig.5B). At day 7, spindle-shaped cells gradually clumped together (Fig.5C). At day 14, cells became aggregates and new islet-like clusters began to appear (Fig.5D). In addition, cell proliferation became slower and islet-like cluster formed on day 21 (Fig.5E).

Pancreas-specific genes expressions
We analyzed the PDX1 gene expression as a key pancreatic development transcription factor and INSULIN gene expression as an endocrine related marker by RT-PCR analysis. Analyses were performed in triplicate on cDNA samples of transduced cells at days 0,7,14 and 21. Our results showed that PDX1 gene expressions increased at day 7 th with a gradual increase at days 14 th and 21 th . (Fig.6, left). As illustrated in figure 6 right the differentiated MSCs at 14 days after induction expressed characteristic INSULIN gene expression which strongly increased at day 21. INSULIN gene expression was detectable at days 14 and 21 of differentiation. Our results showed that PDX1 gene expression was determined earlier.

Viability of mesenchymal stem cells during differentiation into insulin producing cells
The MTT assay was performed on transduced cells at days 0, 7, 14 and 21 post-induction to determine the percentage of cell death during differentiation. Data were from three independent experiments. There was a statistically significant slight reduction (P<0.05) in IPC viability at days 7 and 21 of differentiation compared with undifferentiated day 0 MSCs (Fig.7).

Immunofluorescence analysis
PDX1 and insulin protein were shown in transduced of MSCs PDX1 + using anti-PDX1 and anti-insulin antibodies, which were represented as green dots in the immunofluorescence assay at day 21. Immunofluorescence analysis detected nuclei localization of PDX1 and cytoplasmic localization of insulin. Counter-staining of the nucleus (blue) was performed by DAPI (Fig.8).

Glucose-induced insulin and C-peptide secretion and content
We examined IPCs in response to various glucose concentrations. The insulin level was 3.75 ± 0.70 ng/10 6 cells prior to induction. At a concentration of 5.5 mM glucose, the insulin level was 14.32 ± 1.25 ng/10 6 cells; at 15 mM, it was 22.35 ± 2.3 ng/10 6 cells; and at 25 mM of glucose, the insulin level was 16.1 ± 1.4 ng/10 6 cells (Fig.9A).
The amount of C-peptide secreted was 0.24 ± 0.01 μg/10 6 cells before induction. At a concentration of 5.5 mM, the C-peptide level was 0.89 ± 0.06 μg/10 6 cells, at 15 mM it was 1.53 ± 0.11 μg/10 6 cells and at 25 mM it was 1.30 ± 0.15 μg/10 6 cells (Fig.9B). The intracellular insulin and C-peptide at each concentration of glucose versus cellular total protein are noted on figure 9C and D. Secreted insulin and C-peptide were proportional to glucose concentrations between 5.5-15 mM glucose whereas there was a decrease observed in insulin and C-peptide levels at the 25 mM glucose concentration.

Discussion
Generation of insulin-producing cells from MSCs is new approach to treat type 1 diabetes (11,12). Different cell sources such as cord blood, adipose tissue, and BM have been used to generate IPC (13)(14)(15). BM-MSCs generate more IPCs cells than adipose MSC, because bone marrow MSCs have more islet-like clusters and predominant pancreatic gene expression than adipose MSCs that differentiate into IPC (16).
Our result showed that, MSCs culture produced adherent spindle-like cells and expressed CD90 and CD105 MSC surface markers which confirmed the characterization of these cells. We examined the ability of MSCs to differentiate into adipogenic and osteogenic lineages. We evaluated the potential for BM-MSCs to generate morphologically and functionally pancreatic islet-like structures by genetic manipulation. Our findings revealed that PDX1 made a strong change in gene MSCs and can induced its differentiation to a pancreatic lineage.
Lentiviral vectors are better than adenoviral vectors for gene delivery and therefore higher transgene expression levels have been observed. Lentiviral vectors can transfer the PDX1 gene to MSCs in order to produce IPCs (17,18). Karneili et al. performed differentiated human BM-MSCs in IPCs by over expression of rat Pdx1 gene and they showed expression of islet genes in manipulated BM-MSCs indicated a predisposition for differentiation toward phenotype of islet-cell (19). The homeodomain protein PDX1 is a transcription factors possessing the hallmark 60 amino acid homeodomain that is highly conserved from yeast to human (20). Therefore, the rat Pdx1 gene can differentiate human MSC into IPCs. Transfection by lipofectamine is common in other studies, however we used calcium-phosphate method which generated a sufficient number of transduced cells. Cell-toxicity of the procedure according to the MTT assay showed that mortality at day 7 was not considerable, however significant mortality was observed at days 14 and 21. Our result showed that IPCs released insulin in response to physiological concentrations of glucose. Boroujeni and Aleyasin added B27, nicotinamide and beta fibroblast growth factor (βFGF) to the culture medium in order to promote insulin secretion (21). The current study did not use any chemicals or growth factors in the differentiation protocol. In this study IPCs secreted insulin in response to various concentrations of glucose (0, 5.5, 15, 25 mM) and peak insulin was 23 ng/l at the 15 mM glucose concentration. Peak insulin secretion in other studies was 6.85 ng/l (22), 40 ng/l (21) and 27 ng/l (19). C-peptide in the cell clusters confirmed that insulin release resulted from endogenous synthesis. Our findings showed that insulin was released from IPCs in response to increasing glucose levels up to 15 mM glucose, because the PDX1 activated a number of genes involved in maintaining β-cell identity and function, such as insulin, Glut2 and GK, which suggested that these cells might have a glucose response (23), however, in current study poor response to high concentrations of glucose may be result from lack of expression gene encoding the K + ATP chanel subunits, KIR6.2 and SUR1 (19). In this study, the expression of the genes and protein related IPCs was detected by immunocytochemistry and RT-PCR. The PDX1 gene expressed at the 7 th day of post induction whereas the INSULIN gene expressed at day 14 due to IN-SULIN gene activity-dependent PDX1 gene activity (24). Morphological transformation initiated at day 5 and cells gradually accumulated together at day 14 with the formation of islet like clusters at day 21. The morphological changes in MSCs began from spindle-shaped to round shaped after day 5 post-induction. There were morphological changes to the IPCs at various days of differentiation, which was similar to pancreatic islet cells. The morphology changes was better in our study than other studies (19,21).

Conclusion
Our findings indicate that BM-MSCs can efficiently differentiate into IPCs efficiently by PDX1 over-expression. Lentiviral vectors can deliver the PDX1 gene to MSCs and induce pancreatic differentiation. We have used simple, efficient methods to generate effective IPCs. Additional experiments that include in vivo analyses are needed to confirm our findings.